Effect of Alstonia Boonei Stem Bark Extracts on the Activity of Liver Maker Enzymes in Rats Induced by Ccl4

: This study investigated the outcome of Alstonia boonei stem bark on liver enzymes after inducing the Wistar albino rats with carbon tetrachloride (CCl 4 ). This effect of plant extract was compared with silymarin – a drug commonly used for the treatment of chronic hepatocyte disorder. The plant sample was extracted with ethanol; acute toxicity study of the extract was performed on eighteen Wistar mice, while 30 rats were sacrificed for liver enzymes assay. The rats were divided into six clusters: each cluster has five rats, culster 1 served as control and was given 2 mL/kg b.w - distilled water; clusters 2 – 6 were CCl 4 induced. Cluster 2 was untreated but served as the negative control while cluster 3 was given 0.025 mL or 25kg/kg B.W of Silymarin, which was a regular medicine and aided as the ordinary control. Rats in clusters 4 – 6 were administered - 100, 200 and 500 mg/kg, respectively of ethanol extract for fourteen days. The acute toxicity results of A . boonei extract showed relative fortification due to no death or adversarial responses after 24 hours of the extract administration. A substantial (P ≥ 0.05) surge in ALT action after administering 500 mg/kg proves lesser toxicity was greater dosage. At low dosage of the extract, a non-significant (p ≥ 0.05) rise in AST action specifies that the extract was moderately harmless with no hepatotoxic magnitude at low medication. The substantial reduction of alkaline phosphatase action in cluster 3 rats induced with CCl 4 and given with Silymarin, together with clusters 4 – 6 that were CCl 4 induced and administered with graded doses of A. boonei stem bark extract suggest hepatoprotective properties.


Introduction:
The practice of using specific medicinal plants for the management of definite ailments was beginning to gain recognition. The decrease in efficiency and side effect of artificial medicines and increasing contraindications of their usage/dosages automatically advanced the utilization of beneficial natural plants as drugs. Researches by scientists propose that plant-based drugs and extracts are healthier and safer in managing growing health complications. Therefore, no plant is exclusively unusable to man 1 .
Alstonia boonei is known as "ahun" in the Yoruba language of Nigeria, "ogbu-ora" in the Igbo language of Nigeria and "ukhu" by the Urhobo people of Southern Nigeria 2 . The herbals have several medicinal mixtures including, Nαformylechitamidine, boonein, loganin, lupeol, frolic acid, and b-amyrin including triterpenoids 3 . Over 12 species of Alstonia are dispersed across the world, where two are indigenous to Africa and are known to the traditional healers in West Africa 4  The bark of A. boonei plant is an important traditional medicine in West and Central Africa 6 . Therapeutically, the steam bark holds antirheumatic, anti-inflammatory, and antibiotic properties 7 . It is used in ejecting reserved substance of formation and afterbirth given to lactating women 8 , and applied as a therapy for dysentery, typhoid, gonorrhea, and asthma 6 .
The liver is the prime interior organ -1.5kg in adult human and varies 110 -140g in rats. The liver is also the most energetic and complex organ. Accordingly, the replenishing powers of the liver confer its ability to metabolize, secrete, excrete, store, and decontaminate the body 9 . Notwithstanding its countless interesting tasks, the liver is prone to a lot of damage, caused by various types of liver disorders 10 . This marks the malfunctioning of the liver by hindering its capacity to function well. There are numerous liver disorders such as hepatic cirrhosis, hepatocellular carcinoma, hemochromatosis, autoimmune hepatitis, fulminant hepatitis, and hepatic encephalopathy, which are caused by excessive intake of alcohol, drugs (acetaminophen), and chemicals such as thioacetamide and carbon tetrachloride 11 . This study scrutinized the influence of Alstonia boonei stem bark extracted with ethanol on the liver enzymes after induction of carbon tetrachloride (CCl 4 ) to Wistar albino rats.

Herbal material
The stem bark -Alstonia boonei was utilized. Experimental animals Eighteen 18 mice and thirty albino rats were utilized. Chemicals All chemicals were of analytical grades and are products of British Drug House (BDH).

Gathering of herbal material
The stem bark -A. boonei was obtained from botanical garden at Michael Okpara University of Agriculture, Umudike, Abia State (MOUAUAS), Nigeria, and was authenticated by Dr. K. N. Ibe.

Extract Preparation
The fresh bark -A. boonei was hand-picked from their bole and dried at room temperature. Thereafter, the dried sample was ground into a fine powder, weighed and stored in a sterile container for extraction. The weight of the extract was determined as follows: Volume of extraction (mL)to be administered = (weight of animal (kg) x dose (mg/kg) Concentration of extract (mg/mL) Mining of plant material A 500g of pulverized sample was soaked in 1.5 L of absolute ethanol for 72 hours in a sterile container. The mined plant sample was sieved with a mesh fabric and clarified with What-man No. 1 paper. The deposits were placed in a water bath at 60 o C and allowed to evaporate completely. The extract was weighed to calculate the percentage yield.

Preparation of CCl4
The carbon tetrachloride (CCl 4 ) was mixed with olive oil (2:1 v/v). A 2 mg/kg body weight of carbon tetrachloride was injected intraperitoneally to induced liver failure in rats after 24 hrs fasting before treatment commencement.

Preparation of standard drug
Disinfection of the top of dose vial with 70% alcohol and gauze was carried out. A needle gauge of 24g and 10 mg/kg body weight, for example, a rat that weigh 250 grams, was given 0.025 mL of the standard drug -silymarin. This was drawn into the syringe and injected to the rats. Experimental animal for the study Eighteen mice and 30 albino rats were purchased from the University of Nigeria, Nsukka. The rats were housed at the (MOUAUAS) for seven days involving twelve hours.

Experimental design
The research involved the use 30 male rats and eighteen mice. The eighteen male albino mice remained separated into 2 sets of 9 mice. The Two sets were separated into 3 where each set has 3 mice, for the phase I and phase II to test for acute toxicity, as shown in Tab. 1.

Induction of Carbon tetrachloride
The rats were induced with hepatotoxicity via intraperitoneal injection of 2mg/kg body weight in overnight-fasted animals and allowed for 72 hours without treatment before inducing CCl 4 .

Acute toxicity studies (LD 50 )
The acute toxicity test was determined by the method of 12 . The test was carried out in two phases, with phase I and II were further clustered into three clusters each. Phase I received the extract doses of 10, 100, 200 and 500 mg/kg body weight, respectively while phase II received extract dosages -1000, 2900, and 5000 mg/kg B.W., respectively. Both phases were observed for a period of 24 hours during which they were checked for manifest signs of toxicity, drowsiness, nervousness, incoordination or death. The LD50 was calculated with the geometric mean to the square root of the maximum non-lethal dose and the lowest lethal dose.

Action of Alanine aminotransferase (ALT)
Alanine aminotransferase was assayed using 13 . Alanine aminotransferase was quantified by observing pyruvate hydrazone concentration formed with 2, 4-dinitrophenylhydrazine. The intensity of the color was measured against the blank at 540 nm. A volume, 0.1 mL of serum was pipetted into the tubes. To this, a 0.5 mL of phosphate buffer, Lalanine, and α-oxoglutarate was added. The mixtures were thoroughly mixed and incubated for 30 minutes at 37 o C, and pH 7.4. A 0.5 mL of 2,4dinitrophenylhydrazine was later added into both test tubes while 0.1 mL of the sample was added into the blank test tube. The tubes were incubated for 20 minutes at 25 o C. A 5 mL of NaOH solution was added into each test tube, the mixture was read at 540 nm against the blank after 5 minutes. Aspartate aminotransferase (AST) activity Aspartate aminotransferase activity was assayed according to the method prescribed by 14 . Aspartate aminotransferase is quantify by checking the oxaloacetate hydrazone concentration which formed with 2,4-dinitrophenylhydrazine. The color intensity was quantified together with the blank at 546 nm. A 0.1 mL of serum was pipetted into the test tubes, and a 0.5 mL of reagent 1 was pipetted into the sample and blank tube. The solutions were mixed and incubated for 30 minutes at 37 o C and pH 7.4. A 0.5 mL of reagent-2 -2,4dinitrophenylhydrazine was added, trailed by a 0.1 mL of sample into the blank test tube. The test tubes were mixed and incubated for 20 minutes at 25 o C, while a 5.0 mL of NaOH solution was thereafter added to each test tube and mixed.

Alkaline phosphatase (ALP) activity
The method of 14 was used to assay for alkaline phosphatase activity. The principle lies on the response between alkaline phosphate and phenolphthalein monophosphate (colorless substrate), which gave phosphoric acid and a pink phenolphthalein. A 0.05 mL -distilled water was pipetted into the blank test tube. A 3.0 mL of the substrate was pipetted into each tube and read for the first absorbance -405 nm. Alkaline phosphatase activity was calculated using equation below.

Effects of Alstonia boonei stem bark on alkaline aminotransferase
In cluster 2, rats that were CCl 4 induced and were untreated, together with rats from clusters 4 and 5 which were CCl 4 induced and given 100 and 200 mg/kg b.w of ethanol extract of A. boonei stem bark respectively, showed significantly (P < 0.05) rise in ALT activity. Meanwhile, the rats in clusters 3 and 6 that were CCl 4 induced but given standard drug (Silymarin) and 500 mg/kg b.w of A. boonei stem bark, had no substantial (P > 0.05) upsurge in ALT action likened to normal control (cluster 1) that was not CCl 4 induced. In Fig. 2, it was also observed that cluster 3 that was CCl 4 induced but given Silymarin, and cluster 5 and 6 which were CCl 4 induced but given 200 and 500 mg/kg b.w. A. boonei stem bark showed meaningful (P < 0.05) decrease in ALT compared with cluster 2, although cluster 4 CCl 4 induced but given 100 mg/kg b.w of A. boonei stem bark extract had no major (P > 0.05) decrease in ALT action likened to that of cluster 2 that were CCl 4 induced but untreated. Furthermore, when comparing the ALT action of cluster 3 that was CCl 4 induced but was given Silymarin, it was seen that cluster 4 CCl 4 induced but given 100 mg/kg b.w of A. boonei stem bark exhibited major (P < 0.05) rise, as cluster 5 that was CCl 4 induced but given 200 mg/kg b.w of A. boonei stem bark had no substantial (P > 0.05) increase, while cluster 6 CCl 4 induced but given 500 mg/kg b.w of A. boonei stem bark gave no significant (P > 0.05) decrease in their respective ALT actions.

Effects of Alstonia boonei stem bark extract on Aspartate aminotransferase
The data in Fig. 3 represent the aspartate aminotransferase (AST) activity of carbon tetrachloride (CCl 4 ) induced male Wistar albino rats. Results showed that cluster 2 that was CCl 4 made but was not given the extract, cluster 3 that was CCl 4 made but was given Silymarin, together with cluster 5 and 6 that were CCl 4 made but were given 200 and 500 mg/kg b.w of A. boonei stem bark, had a substantial (P < 0.05) increase in AST action, whereas cluster 4 that was CCl 4 made but was given low dose of A. boonei stem bark revealed no major (P > 0.05) rise in AST action relative to cluster 1 not CCl 4 induced. However, it was detected that the AST activities of rats in cluster 3 that were CCl 4 induced but given standard drug -Silymarin and cluster 4 -6 that were CCl 4 made but were given 100, 200 and 500 mg/kg b.w A. boonei stem bark extract, separately revealed substantial (P < 0.05) decrease relative to cluster 2 that was CCl 4 induced and were not given. The AST activities of the rats in cluster 3 that was CCl 4 induced but given -Silymarin was compared with that of cluster 4 that was CCl 4 induced but given 100 mg/kg b.w of A. boonei stem bark, there was a substantial (P < 0.05) decrease, as the AST action of cluster 5 that was CCl 4 induced but was given 200 mg/kg b.w of A. boonei stem bark exhibited no substantial (P > 0.05) increase, despite the fact cluster 6 that was CCl 4 induced and given 500 mg/kg body weight of A. boonei stem bark revealed no significant (P > 0.05) decrease in AST action.  Fig. 4 illustrates the alkaline phosphatase (ALP) activity of male rats induced with CCl 4 . The ALP action of cluster 1 induced with CCl 4 was compared with other clusters, it was witnessed that only cluster 2 rats CCl 4 induced but untreated showed substantial (p < 0.05) increase in ALP action, while cluster 3 induced with CCl 4 but given Silymarin and clusters 4 -6 CCl 4 induced but given classified dosages of A. boonei stem bark extract showed substantial reduction in their respective ALP activities. Nevertheless, it was perceived that the ALP activities of rats in cluster 3 that was CCl 4 induced but given Silymarin, and cluster 4 -6 that was CCl 4 induced and given 100, 200 and 500 mg/kg body weight of A. boonei stem bark extract, showed substantial (P < 0.05) decrease corresponding with cluster 2 that was CCl 4 induced but untreated. Rats in cluster 4 that were CCl 4 induced but given 100 mg/kg b.w of A. boonei stem bark had no significant (P > 0.05) reduction in ALP activity, as cluster 5 that was CCl 4 induced but given 200 and 500 mg/kg b.w of A. boonei stem bark showed substantial reduction in ALP actions compared to ALP activity of cluster 3 that was CCl 4 induced but was given Silymarin only.

Discussion
The study evaluated the outcome of Alstonia boonei stem bark extracted with ethanol on the liver enzymes and hematological properties after inducing carbon tetrachloride (CCl 4 ) in male Wistar albino rats. The liver is the main organ for metabolism and detoxification of drugs as well as environmental chemicals 15 . The liver cells are unprotected against significant concentrations of chemicals, thus, the function of the liver may be affected adversely 16 . The impairment to the liver could lead to elevated levels of liver enzymes and their activities in the extrahepatic tissues that may ultimately lead to the development, progression, and complication of various hepatic disorders. There are several conditions of liver disorders that can be induced by different means such as alcohol liver disease, fatty liver disease, hepatitis and several others 17 . The no death or adversarial behavior saw in the LD 50 study of A. boonei stem bark extract suggests safeties at a high dose of 500 mg/kg body weight. The effect was ascribed to the moderately non-toxic or that the extract possesses low toxicity potential. However, most substances may be chronically toxic in that they may not be toxic but manifest signs of toxicity on continued consumption. The absence of adverse behavior may also be attributed to the extract containing a very low concentration of possible toxicants, with lack of sufficient concentration and interval for the expression of toxicity.
Alanine aminotransferase (ALT) is a liverspecific enzyme used in identifying liver damage as it is rarely seen except in liver disorder 18 . The substantial rise in ALT activity in all the rats induced with CCl 4 but untreated; together with those induced but given with a small amount of the ethanol extract indicate toxicity and potential liver damage. This might be as a result of the outflow of the enzyme into the extrahepatic surroundings caused by a compromised plasma membrane resulting in the continuous administration of the extract. The upsurge in ALT action detected, corroborate with the findings of 19 , who reported that increased activities of ALT are associated with hepatitis and other liver disorders. The nonsignificant (P > 0.05) upsurge in ALT action saw in cluster 3 CCl 4 induced but given standard drug (Silymarin) as well as cluster 6 that were CCl 4 induced but were given 500 mg/kg b.w of A. boonei stem bark was credited to the hepatoprotective effects of A. boonei stem bark, this implied that the extract may not be very toxic or have lesser toxic impact at a high prescription of 500 mg/kg b.w, and may not have affected the ALT production. Thus, administration of Silymarin and a high dose of the extract respectively caused the liver to gradually recover from the damage caused by CCL 4 induction, hence preventing leakage of ALT to extrahepatic tissues as indicated by a decrease in its activity. Aspartate aminotransferase catalyzes the reductive transfer of an amino group from aspartate to 2-oxo-glutarate to produce oxaloacetate and glutamate. It helps in detecting the hepatocellular disorder, at the same time; it is well-thought-out a less specific biomarker-enzyme for hepatocellular damage 20 . The non-substantial rise in AST action of clusters that received a low dose of A. boonei extract shows moderate safety with no hepatotoxic outcome at low dose. However, the substantial rise in AST action in untreated clusters that were given standard drug and those administered high categorized dosages, suggest that the extract may possess hepatotoxic outcome at high concentrations. Also, a rise in AST action may be due to the liver damage 21 .
The substantial reduction in alkaline phosphatase action in cluster 3 rats that were CCl 4 induced but given with the standard drug (Silymarin), together with clusters 4 -6 that were CCl 4 induced but administered graded doses of A. boonei stem bark suggest the extract possess hepatoprotective properties 22 . In other words, a noticeable (P < 0.05) upsurge in ALP action of rats that were CCl 4 induced but untreated may be ascribed to CCl 4 action on the liver that impaired the liver integrity. These toxic influence may lead to membrane and liver cells damage occasioned by a rise in permeability of the liver membrane causing an elevation in ALP action at the extrahepatic cell.

Conclusion:
The administration of 500 mg/kg body weight for acute toxicity study shows that the plant is less toxic as no death was recorded. The Alstonia boonei stem bark influence the activity of the liver enzymes in a dose-dependent perspective. This suggests that at high dosage, the activities of the liver enzymes is greatly regulated, conferring its capacity to protect the hepatocytes against injuries.